Protection from infectious agents known to be major causes of death worldwide, such as influenza, tuberculosis, and malaria, as well as potential release of bioweaponized agents which cause plague, tularemia, and meilloidosis require vaccines that generate humoral and T-cell responses. Effective component vaccines require the addition of adjuvants to increase their immunogenic capacities. Until recently alum salts, which require repeated applications and tend to be skewed towards T helper TH2-based immunity (humoral) rather than TH1, (cellular) were the only adjuvants approved for use in human vaccines. Recently, the lipid A mimetic (monophosphoryl lipid A, MPL) adjuvant has been combined with alum (AS04) in two FDA-approved vaccines (Cervarix (Human Papilloma Virus), and Fendrix (Hepatitis B Virus)). Additionally, synthetic lipid A mimetics aminoalkyl glucosaminide phosphates (AGPs) that also signal through Toll-Like Receptor 4 (TLR4) are being studied as both adjuvants or stand-alone immunogenic compounds. Thus, TLR4 agonists show great promise for use as adjuvants in component vaccines. However, the approved TLR4 agonist, MPL has distinct deficiencies both in potency and structural consistency and AGPs are labor intensive and costly to synthesize. Therefore, the goal of this revised R21 application is to undertake a novel approach of using Bacterial Enzymatic Combinatorial Chemistry (BECC) to make rationally-designed lipid A structures by modifying the lipid A structure of the lipooligosaccharide (LOS) from an attenuated (BSL-2 approved) Yersinia pestis (Yp) strain. This approach will use the non-stimulatory, hypoacylated, and bisphosphorylated lipid A structure present in LOS synthesized by this Yp strain as a scaffold to be modified by heterologous in trans expression of lipid A biosynthesis enzymes obtained from a wide variety of bacterial backgrounds with specificities for the removal or addition of fatty acid chain, phosphates moieties, and carbohydrates to the lipid A backbone, This process allow for the safe, cost effective, and efficient design of molecules with immunostimulatory potential. We will test the immunotherapeutic potential of these new molecules in vitro and in vivo to identify novel molecules representing adjuvants and/or immunomodulating reagents. We will also include well-characterized immunostimulants, such as MPL and known LPS structures as comparisons to the molecules synthesized by BECC. The successful demonstration of protective innate/adaptive immune responses by this novel approach of creating new adjuvants, in highly relevant in vivo animal model(s) could have important implications in the field of antigen recognition, formulation, and vaccine design.